CBS, a central enzyme in the transsulfuration pathway, plays an essential role in homocysteine (Hcy) metabolism in eukaryotes (Mudd et al., 2001, in THE METABOLIC AND MOLECULAR BASES OF INHERITED DISEASE, 8 Ed., pp. 2007-2056, McGraw-Hill, New York). CBS catalyzes Hcy condensation with L-serine to form cystathionine. When CBS activity is dramatically reduced or absent, as a result of certain genetic mutations, Hcy builds up in tissues and blood. The CBS enzyme catalyzes a pyridoxal-5′-phosphate (PLP; Vitamin B6)-dependent condensation of serine and homocysteine to form cystathionine, which is then used to produce cysteine by another PLP-dependent enzyme, cystathionine γ-lyase. In mammalian cells that possess the transsulfuration pathway, CBS occupies a key regulatory position between the remethylation of Hcy to methionine or its alternative use in the biosynthesis of cysteine.
In healthy normal individuals, CBS-mediated conversion of Hcy to cystathionine is the rate-limiting intermediate step of methionine (Met) metabolism to cysteine (Cys). Vitamin B6 is an essential coenzyme for this process. In patients with certain genetic mutations in the CBS enzyme, the conversion of Hcy to cystathionine is slowed or absent, resulting in elevations in the serum concentrations of the enzymatic substrate (Hcy) and a corresponding decrease in the serum concentrations of the enzymatic product (cystathionine). The clinical condition of an elevated serum level of Hcy, and its concomitant excretion into the urine, is collectively known as homocystinuria.
The estimates on the prevalence of homocystinuria vary widely. Data from newborn screening and clinical ascertainment provide a range of 1:200,000 to 1:335,000 live births (Mudd et al., 2001). Recent evidence from DNA screening studies of newborns in Denmark, Germany, Norway and the Czech Republic found that the true incidence may be as high as ˜1:6,000 (Gaustadnes et al., 1999, N Engl J Med. 1340:1513; Linnebank et al., 2001, Thromb Haemost. 85:986; Refsum et al., 2004, Clin. Chem. 50:3; Sokolova et al., 2001, Hum Mutat. 18:548). Additionally, recent work has indicated that CBSDH patients exist with either psychiatric or cardiovascular complications but are currently undiagnosed due to a lack of the characteristic connective tissue defects that are typically instrumental in diagnosis (Li and Stewart, 1999, Pathol. 31:221; Linnebank et al., 2003, J. Inherited Metabol. Dis. 26: 509; Maclean et al., 2002, Hum Mutat. 19:641). The primary health problems associated with CBS-deficient homocystinuria (CBSDH) include: cardiovascular disease with a predisposition to thrombosis, resulting in a high rate of mortality in untreated and partially treated patients; connective tissue problems affecting the ocular system with progressive myopia and lens dislocation; connective tissue problems affecting the skeleton characterized by marfanoid habitus, osteoporosis, and scoliosis; and central nervous system problems, including mental retardation and seizures.
The therapeutic resolution of CBS-associated homocystinuria is dependent upon the type of mutations present in the CBS gene. Approximately 160 pathogenic mutations of the CBS gene have been identified to date in humans. There is a functional trichotomy in the nature of pathogenic mutations associated with CBSDH. One group of mutations is classified as “pyridoxine-responsive,” where CBS enzyme function can be restored by high dose Vitamin B6 therapy. This treatment can be effective, but does not always mitigate the pathological events in these patients, and some of the events occur even in these patients over time. The second group of functional mutations is represented by the “C-terminal CBS mutants” that are defective in their ability to respond to post-translational up-regulation by S-adenosylmethionine. Patients with this class of mutations usually lack the mental retardation and connective tissue aspects of the phenotype. This class is detected after measurement of plasma Hcy levels following an idiopathic thrombotic event before the age of 40 years (Maclean et al., 2002, Hum Mutat. 19: 641-55). The final group of CBSDH mutations is “classical homocystinuria,” which represents the most severe form of the disease. For these latter two groups of patients, Vitamin B6 therapy in isolation does not effectively lower serum Hcy levels.
The pathophysiology of homozygous CBS deficiency is undoubtedly complex, but there is a consensus that the fundamental instigator of end-organ injury is an extreme elevation of serum Hcy. The toxicity of profound elevations in blood and tissue concentrations of Hcy may ensue from the molecular reactivity and biological effects of Hcy per se or from its metabolites (e.g., Hcy-thiolactone) that affect a number of biological processes (Jakubowski et al., 2008, FASEB J 22: 4071-6). Abnormalities in chronic platelet aggregation, changes in vascular parameters, and endothelial dysfunction have all been described in patients with homocystinuria.
Currently, three treatment options exist for the treatment of CBSDH:                1) Increase of residual activity of CBS activity using pharmacologic doses of Vitamin B6 in Vitamin B6-responsive patients;        2) Lowering of serum Hcy by a diet with a strict restriction of the intake of Met; and        3) Detoxification by betaine-mediated conversion of Hcy into Met, thus lowering serum Hcy concentration.Each of these three therapies is aimed at lowering serum Hcy concentration. The standard treatment for individuals affected with Vitamin B6 non-responsive CBSDH consists of a Met-restricted diet supplemented with a metabolic formula and Cys (which has become a conditionally essential amino acid in this condition). Intake of meat, dairy products and other food high in natural protein is prohibited. Daily consumption of a poorly palatable, synthetic metabolic formula containing amino acids and micronutrients is required to prevent secondary malnutrition. Supplementation with betaine (tradename: CYSTADANE, synonym: trimethylglycine) is also a standard therapy. Betaine serves as a methyl donor for the remethylation of Hcy to Met catalyzed by betaine-homocysteinemethyltransferase in the liver (Wilcken et al., 1983, N Engl J Med 309, (8), 448-53). Dietary compliance generally has been poor, even in those medical centers where optimal care and resources are provided, and this noncompliance has major implications on the development of life-threatening complications of homocystinuria.        
The evidence described in the above sections is summarized in the following points:                Untreated homocystinuria has a high rate of complications in the vasculature, connective tissue, and central nervous system.        Treatments that lower serum Hcy, such as a severely Met-restricted diet and betaine, lower the associated clinical problems if executed well. Improvements in cognitive performance require treatment initiation in early infancy.        Compliance with diet is uniformly poor. In patients initiating therapy in the neonatal period, the loss of compliance occurs in adolescence. In patients beginning therapy after the neonatal period, compliance is abysmal at all ages. The extreme difficulties of the current treatment approach are most readily evident in patients who experience life-threatening symptoms preventable by diet, and yet are unable to adhere to this treatment.        Failure of dietary compliance causes increased serum Hcy, resurgence of complications in the vascular and connective tissue including fatal and incapacitating events, and risks severe side-effects such as cerebral edema (from excessive serum Met concentration) or severe malnutrition (from lack of essential amino acids).        The most effective therapeutic strategy is to increase enzyme activity, as is evident when given pyridoxine to Vitamin B6 responsive homocystinuria. This strategy is not possible for Vitamin B6 non-responsive patients due to the mutation status, and increased enzyme activity in these patients will depend on the delivery of exogenous enzyme, i.e. enzyme replacement therapy (ERT) (a strategy that has never been attempted for treating homocystinuria).        Of the three current treatment strategies with demonstrated efficacy: (1) increasing enzyme activity with pyridoxine in Vitamin B6 responsive patients; (2) reducing accumulating metabolites by a Met-restricted diet; and (3) detoxification by enzyme activity of the betaine-homocysteine methyltransferase in betaine treatment, all have in common a lowering of total Hcy in plasma (Walter, et al., 1998, Eur J Pediatr 157(Suppl 2): S71-6).        
In addition, for all current treatment strategies (except B6 supplementation, which is appropriate for only a subset of homocystinurics), reduction in homocysteine is not accompanied by increases in cystathionine or cysteine. Because it has not been established that excess homocysteine, rather than a deficiency of a downstream metabolite, is responsible for clinical symptoms, treatment strategies that are limited to reducing homocysteine may be inadequate for providing robust and effective treatment options.
Thus, there remains a need in the art for more effective treatment strategies for patients with homocystinuria.